BRITISH MEDICAL JOURNAL VOLUME 285 4 DECEMBER 1982 1601

pathy. Only one abnormal ratio was found in the 75 diabetics ofgrades 0 and 1, in whom autonomic function was normal on conven-tional testing. In grade 2, 41 of the 47 diabetics gave abnormal orborderline results. The ratios in the 12 with symptomatic autonomicneuropathy (grade 3) were all well below the normal range.

Repeat measurements of the E:I ratio varied little; the coefficientsof variation were 8 9° in healthy subjects and 5 3%/ in diabetics.

Discussion

Measurement of variation in heart rate during deep breathingis generally agreed to be a sensitive index of autonomic dysfunc-tion, comparing favourably with tests based on changes inposture.1 3 Adaptation of a repeated deep breathing test for usewith a conventional electrocardiogram to obtain an E:I ratiowas first described by Sundkvist et al,11 which represented aconsiderable simplification in technique over previous methods.These authors did not define the influence of age on this ratiobut set the lower limit of normal at an E:I ratio of 1-1. Whenapplied to the ratios measured here this limit gave three false-positive and 16 false-negative diagnoses. The age-related normalrange described in this report increases the diagnostic value ofthe E:I ratio test since there were only one false-positive and sixfalse-negative results.

There does not appear to be any advantage in averaging thevariations in heart rate from a sequence of forced respirationscompared with measuring the change resulting from a singledeep breath. The change in rate is greater on the first breath1 12and, in contrast to the mean, is not affected by the resting heart-rate,5 which simplifies the construction of a normal range.

I conclude, therefore, that the E:I ratio test of sinus ar-rhythmia is an accurate and reliable method of screeningdiabetic patients for autonomic dysfunction. It may be per-formed rapidly by a nurse or technician with any electrocardio-

graphic apparatus and immediate reference made to the normalrange. It is thus appropriate for use in the diabetic clinic.

I am grateful to the British Diabetic Association for financialsupport, Professor S E Smith for statistical advice, and Professor P HSonksen and Dr C Lowy for allowing me to study their patients.

References

Mackay JD, Page MMcB, Cambridge J, Watkins PJ. Diabetic autonomicneuropathy. The diagnostic value of heart rate monitoring. Diabetologia1980;18 :471-8.

2 Ewing DJ, Borsey DQ, Bellavere F, Clarke BF. Cardiac autonomicneuropathy in diabetes: comparison of measures of R-R intervalvariation. Diabetologia 1981 ;21 :18-24.

3Anonymous. Diagnosis of autonomic neuropathy. (Editorial.) Br Med J1978;ii :910-1.

4Wheeler T, Watkins PJ. Cardiac denervation in diabetes. Br MedJ3 1973;iv :584-6.

5Smith SE, Smith SA. Heart rate variability in healthy subjects measuredwith a bedside computer-based technique. Clin Sci 1981;61:379-83.

6 Wieling W, van Brederode JFM, de Rijk LG, Borst C, Dunning AJ.Reflex control of heart rate in normal subjects in relation to age: a database for cardiac vagal neuropathy. Diabetologia 1982;22:163-6.

7 Smith SE, Smith SA, Brown PM, Fox C, Sonksen P. Pupillary signs indiabetic autonomic neuropathy. Br MedJ' 1978;ii:924-7.

8 Smith SA, Smith SE. Evidence for a neuropathic aetiology in the smallpupil of diabetes mellitus. Br J Ophthalmol (in press).

9 Baker RT, Nelder JA. The GLIM system manual for release 3. Oxford:Numerical Algorithms Group, 1978.

0 Fisher RA. Intraclass correlations. In: Statistical methods for researchworkers. 14th ed. Edinburgh: Oliver and Boyd, 1970:217-49.

1' Sundkvist G, Almer L-O, Lilja B. Respiratory influence on heart rate indiabetes mellitus. Br MedJ3 1979;i:924-5.

12 Bennett T, Farquhar IK, Hosking DJ, Hampton JR. Assessment ofmethods for estimating autonomic nervous control of the heart inpatients with diabetes mellitus. Diabetes 1978;27:1167-74.

(Accepted 16 September 1982)

Microscopic colitis-a cause of chronic watery diarrhoea

JEREMY G C KINGHAM, DAVID A LEVISON, JOANNA A BALL, ANTHONY M DAWSON

Abstract

Six patients with severe watery diarrhoea were foundto have microscopic total colitis. None had any ab-normality detectable by conventional tests used todiagnose inflammatory bowel disease-namely, bariumradiology and endoscopy. The diagnosis could only bemade by microscopic examination of biopsy specimensfrom the apparently normal colon. Anaemia, raisederythrocyte sedimentation rate, hypokalaemia, andhypoalbuminaemia were common findings. Small-bowel function was normal in all, though three patientshad jejunal lesions of uncertain relevance but seeminglyunrelated to the diarrhoea. The five patients givenanti-inflammatory drugs showed a satisfactory response

Departments of Gastroenterology and Pathology, St Bartholomew'sHospital, London ECIA 7BE

JEREMY G C KINGHAM, MD, MRCP, senior medical registrarDAVID A LEVISON, MB, MRCPATH, senior lecturerJOANNA A BALL, MB, BS, house physicianANTHONY M DAWSON, MD, FRCP, consultant physician

with improvement of the diarrhoea and colonic in-flammation and return to normal of the abnormallaboratory findings.Microscopic colitis is responsible for a proportion of

cases of intractable diarrhoea of 'obscure origin andrectal and colonic biopsies should be undertaken insuch cases.

Introduction

Occasionally patients with large-volume diarrhoea undergoextensive investigations with no cause found. We describe sixsuch patients seen over the past 10 years in whom biopsy of anapparently normal colon showed microscopic colitis and led tosatisfactory treatment.

Patients and methods

All patients were referred for inpatient investigation of diarrhoeaafter negative findings on investigations at other hospitals. All werewhite and none had lived abroad. Further investigations excluded (asfar as this is possible) diarrhoea induced by tumour or laxatives. No

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patients had any abnormality on the findings of conventional testscurrently used to diagnose inflammatory bowel disease-namely,sigmoidoscopy, colonoscopy, barium enema, and small-bowel meal.The tables give details of the patients.

CLINICAL FEATURES

In all patients diarrhoea was the main complaint, with maximumdaily stool volumes from 900 to 2100 ml. Not every patient haddiarrhoea on every day during time in hospital, some passing normalvolumes of semiformed stool on occasions, but the mean documenteddaily stool volume exceeded 500 ml in all cases (normal 50-250 ml).Table I shows the main clinical features. Four of the six patients

were women. Age ranged from 39 to 62 years and diarrhoea had beenpresent for from three months to 25 years at the time of diagnosis.The stools were loose or watery and pale to dark brown but withoutblood. The average daily frequency of bowel movement was six. Allpatients had urgency of defecation and nocturnal diarrhoea; fourwere occasionally incontinent. All had some associated abdominaldiscomfort and malaise,'though none suffered severe pain. Weightloss of more than 7 lb (3-2 kg) was noted in five, and ankle oedemain two. One patient was known to have coeliac disease, though was

in remission on gluten-free diet. All patients had previously triedsymptomatic remedies for their diarrhoea without noticeable benefit.The father of one patient had colitis, while her two siblings hadjuvenile rheumatoid arthritis.No specific features were noted on physical examination though

weight loss was obvious in two. No perianal disease other thanexcoriation secondary to diarrhoea was seen.

FINDINGS ON ENDOSCOPY AND RADIOLOGY

Sigmoidoscopy was performed on several occasions in all patientsand total colonoscopy at least once. These examinations gave un-

equivocally normal results. High-quality double-contrast bariumenema was performed on all patients and more than' once in five ofthe six. None showed any of the changes of inflammatory boweldisease but one showed sigmoid diverticulosis and in one the colonwas rather dilated (diameter 5 cm) though the haustral pattern andmucosal outline were normal. Barium meal and small-bowel mealexaminations were performed separately at least once in all patients.No mucosal abnormalities were seen, though in three the transit timefrom mouth to caecum was rather prolonged (greater than six hours)and in two of these the jejunum was considered slightly dilated. Ofthese two, one (case 2) had subtotal villous atrophy. There was nosuggestion of inflammatory bowel disease in any of the bariumstudies of the upper intestinal tract.

HISTOLOGICAL FINDINGS

Rectum and colon-Microscopic colitis was diagnosed histologicallyas the colon was macroscopically normal. All the rectal and colonicbiopsy specimens (31 in all) from these six patients were abnormalwith diffuse,-mild but definite chronic inflammation of the laminapropria (fig 1). The inflammation was composed predominantly ofplasma cells with various admixtures of lymphocytes, macrophages,eosinophil and neutrophil polymorphs. No granulomata were seen.There was only minor distortion of the glands, and goblet cells were,at most, only slightly reduced in numbers. The inflammation wasfound to be remarkably uniform, indicating total colitis. Biopsyspecimens taken after starting treatment with sulphasalazine showedfewer inflammatory cells in the lamina propria, though in none was

BRITISH MEDICAL JOURNAL VOLUME 285- 4 DECEMBER: 1982

inflammation completely abolished. In one patient (case 4) there was

slight fibrous thickening of the epithelial basement membrane,though this was much less than that described in so-called collagenouscolitis.1 2 The possibility that this degree of inflammation might besecondary to the diarrhoea was tested by comparing the biopsyspecimens from these six patients with rectal and colonic biopsyspecimens from other patients with chronic diarrhoea of comparableseverity and duration. Some of these control patients had diarrhoeaof known ca'use (ileal Crohn's disease, vipoma) while others had

FIG 1-Rectal biopsy specimen from patient with microscopic colitis showingmoderate diffuse chronic inflammation of the lamina propria and minimalgoblet cell depletion. Haemotoxylin and eosin x 100 (original magnification).

FIG 2-Rectal biopsy specimen without appreciable histological abnormalityfrom a patient with chronic diarrhoea, still undiagnosed but not, by defini-tion, microscopic colitis. Haemotoxylin and eosinX 100 (original magni-fication).

TABLE I-Clinical details of six patients studied

Volume of stools/day Frequency of stools/day and consistencyCase No Sex Age at presentation Duration of diarrhoea

Before treatment After treatment Before treatment After treatment

1 M 41 1 year 100-1000 60-400 3 watery 2 soft2 F 62 25 years 200-1200 130-500 12 watery 3 soft (occasionally loose)3 F 56 1 year 250-1700 100-350 6 watery 2 formed (occasionally loose)4* F 54 20 years 400-900 6 watery 2 soft5 F 61 4 years 1000-2100 150-350 8 watery 2 firm (occasionally loose)6* M 53 3 months 800-1100 5 loose/watery 4 loose/watery

*Case 4 was unable to tolerate sulphasalazine and case 6 refused treatment.

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diarrhoea the cause of which was undiagnosed. The biopsy specimensfrom these control cases were all reported as normal when reviewedby one of us (DAL) and were identified as normal without hesitationwhen reviewed blind, mixed in with the microcolitic cases, by anotherpathologist (Dr A G Stansfeld) (fig 2).

Jtejunum and ileum-All six patients had at least one jejunal biopsy.In three patients these specimens were abnormal, the histologicalfeatures showing subtotal villous atrophy in one, treated coeliac diseasein one (mild partial villous atrophy and a slight excess of chronicinflammatory cells in the lamina propria), and mild non-specificinflammation in one. Further details of these three patients are givenin the case histories. Three patients had ileal biopsies, which gavenormal results.

LABORATORY INVESTIGATIONS

Table II gives the principal abnormalities found. Four had anaemia;iron deficient in two, normochromic or normocytic in two; and infour the erythrocyte sedimentation rate was greater than 40 mm inthe first hour. Four had low serum albumin concentrations and fourhad hypokalaemia during an attack of the diarrhoea. All the haemato-logical and biochemical abnormalities returned to normal onceremission had been induced.Table III shows the investigations with essentially negative findings

undertaken in the search for a cause of the diarrhoea. One patient(case 3) had been on a gluten-free diet for five years at thetime of investigation. It is clear that standard tests of renal, liver, andabsorptive function gave essentially normal results.

Findings of hormone screens were normal in all cases. There wasno evidence of stool pathogens or small-bowel bacterial overgrowth.Tests of pancreatic exocrine function (in five) and gastric acidsecretion (in four) gave normal results. Jejunal perfusion studies inthree patients showed normal absorption of sugar, amino-acids,water, and electrolytes.

In all patients purgative abuse was considered. To the best of ourability this was excluded by testing stools for phenolphthalein andurine for anthraquinones, and by careful search of the patient'spersonal belongings, bed, and bedside locker.

TABLE II-Principal abnormalities found in the six patients

1 2 3 4 5 6

Haemoglobin (g/dl) 7-3 13 8 11-0 10-2 9-7 13-8Erythrocyte sedimentation rate (mm in

first hour) 43 77 47 23 43 5Albumin (g/1) (normal range 36-48 g/l) 35 28 35 43 31 36Potassium (mmol/1) (normal range 3-5-

5 0 mmol/) .. 4-4 2-3 2-1 4-0 3-3 3-0

Conversion: SI to traditionial units-Potassium = 1 mmol/l 1 mEq/l.

TABLE III-Results of investigations undertaken in search for cause of diarrhoea

1 2 3 4 5 6

Plasma urea and electrolyte (excluding potassiumions) .. N N N N N N

Serum liver function tests . . N N N N N NSerum calcium and magnesium ions N N N N N NSerum iron (Iimol/l) . . 30 N N N 4-0 NTotal iron binding capacity (,umol/l) 75 N N N 63 NSerum vitaminB .. N N N N N NSerum and red cell folate . . N N N N N NSerum globulins ..N N N N N NSerum IgG. IgA, IgM, and autoantibodies N N N N N NHormone screen* .N N N N N NStool microscopy and culture . . N N N N N NJejunal juice microscopy and culture - - N - N -24-hour faecal fat .N N N N N NCarbon 14 bile salt breath test . . N N N N N NSchilling test ( excretion in 24 h) N N N N 5 % NXylose tolerance test . . N N N -. N -Lundh test .N N N - N NJejunal perfusion . . - N N N - -Gastric acid secretion . . N - N - - NPurgative screen . . N N N N N NIleal biopsy.. .- - N - N N

*Hormone screen-Gastrin, 5-hydroxyindole-acetic acid, thyroid function in allcases. Calcitonin, vasoactive intestinal polypeptide cases 1, 2, 4, 5, 6. Glucagon,somatostatin, neurotensin cases 4, 5, 6.N = Normal.

Conversion: SI to traditional units-Iron: 1 Uumol/l z 6 ,ug/100 ml. Total ironbinding capacity: 1 ,umol/l v6 jig/100 ml.

1603

CASE HISTORIES

Three patients had abnormal findings on jejunal biopsy.Case 2 presented with a long history of watery diarrhoea, un-

associated with malabsorption (see table III). She was shown tohave both microscopic colitis and subtotal villous atrophy. She atea normal diet and was treated with sulphasalazine alone. which ledto rapid clinical improvement and the return of the erythrocytesedimentation rate and serum potassium concentrations to normal.Six months later repeat colonic and jejunal biopsies showed consider-able improvement.

Case 3-This woman had presented initially with steatorrhoea,folate deficiency, and xylose malabsorption typical of coeliac disease.Jejunal biopsy confirmed subtotal villous atrophy. Clinical, bio-chemical, and histological remission of the coeliac disease wasachieved within three months of starting gluten-free diet and has beenmaintained since. Her symptoms of colitis started some five yearsafter the coeliac disease had been successfully treated. The diarrhoeawas quite different, being profuse and watery without fat. She wastreated with sulphasalazine 4 g a day with considerable improvement.She remained on gluten-free diet throughout.

Case 5 had a three-year history of copious watery diarrhoea.Investigations showed both a microscopic colitis and a slightlyabnormal jejunal biopsy specimen (minimal villous stunting andsubmucosal inflammation but normal epithelium). The changes were

non-specific and did not suggest coeliac disease. There was no fat orfolate malabsorption but B12 absorption was somewhat impaired(see table III). Treatment with steroids and sulphasalazine resultedin clinical, biochemical, and histological remission. Thereafter sheremained well taking sulphasalazine alone.

RESPONSE TO TREATMENT

Five patients responded rapidly to treatment with anti-inflammatoryagents used in inflammatory bowel disease. In two (cases 1 and 5)oral prednisolone and sulphasalazine were given initially and thereaftersulphasalazine alone (3-4 g/day) as maintenance. In two others(cases 2 and 3) sulphasalazine alone was used successfully (3 g/day).One further patient (case 4) was unable to tolerate sulphasalazinebecause of gastrointestinal side effects. She was given indomethacin100 mg/day with considerable improvement. The remaining patient(case 6) declined treatment and his condition is unchanged on

follow-up.

Discussion

These six cases showed symptomatic colitis in the absence ofendoscopic and x-ray changes. The diagnosis could only bemade by biopsy and microscopy. The nature of this micro-scopic colitis has yet to be determined. The clinical picturedoes not suggest a close relation to ulcerative or Crohn'scolitis: the severity of the diarrhoea would be quite atypical inconventional inflammatory bowel disease in the absence ofgross disease and bleeding. Histologically, the uniform distri-bution of the colitis would be more akin to ulcerative thanCrohn's colitis but there were no other histological featuresparticularly suggestive of this or of infective or ischaemiccolitis.

Microscopic colitis appears to be a clinical entity quiteseparate from "minimal change colitis"3 in which, despitenormal appearances on sigmoidoscopy and largely normalfindings of barium enema, overt changes of Crohn's colitisproximal to the rectum were seen at colonoscopy. Extensiveinvestigations in our six patients failed to reveal any otherpossible cause of diarrhoea, with the exception that three hadabnormal jejunal biopsies. The importance of these jejunallesions is unclear but they cannot reasonably account for thediarrhoea in the face of normal results of tests of absorptivefunction including jejunal perfusion. Indeed, this pattern ofprofuse watery diarrhoea would be rare even with severe

malabsorption. Although occasional reports of coeliac diseaseand colitis in the same patient have appeared,4 the frequencysuggests a chance association. There seems to be a commoner

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association between overt ulcerative colitis and less specificjejunal abnormalities6-8 whose severity reflects the clinicalactivity of the colitis.

Microscopic colitis is not generally recognised, thoughRead et a19 reported minor histological changes in the colondescribed as microscopic colitis in eight of 27 patients investi-gated for intractable diarrhoea. No conclusions, however, weredrawn from this finding.The possibility that surreptitious laxative ingestion caused the

diarrhoea and microscopic colitis in our patients cannot betotally disproved. Nonetheless, we consider it unlikely as wefailed to find laxatives in stool, urine, or personal belongings ofthe patients; melanosis coli was not found endoscopically andhistologically; the histological changes in our cases differedfrom those described in the cathartic colon10 11; and, most im-portantly, patients responded favourably to sulphasalazine.We conclude that microscopic colitis is responsible for a

proportion of cases of intractable diarrhoea of obscure originand may respond to treatment with anti-inflammatory drugs.Rectal and colonic biopsies are mandatory in the investigationof such patients even though findings of colonic radiology andendoscopy are normal.We thank Miss Helen Trussler for typing the manuscript.

References

ILindstrom CG. "Collagenous colitis" with watery diarrhoea-a newentity? Pathologica Europa 1976;1 1:87-9.

2 Teglbjaerg PS, Thaysen EH. Collagenous colitis: an ultrastructuralstudy of a case. Gastroenterology 1982 ;82 :561-3.

3 Elliot PR, Williams CB, Lennard-Jones JE, et al. Colonoscopic diagnosisof minimal change colitis in patients with a normal sigmoidoscopy andnormal air contrast barium enema. Lancet 1982;i:650-1.

4Falchuk KR, Falchuk ZM. Selective IgA deficiency, ulcerative colitis andgluten sensitive enteropathy-a unique association. Gastroenterology1975 ;69 :503-6.

Kumar PJ, O'Donoghue DP, Gibson J, Stansfeld A, Dawson AM. Thecoexistence of inflammatory bowel lesions in coeliac disease. PostgradMedJ 1979;55:753-6.

6 Salem SN, Truelove SL. Small intestinal and gastric abnormalities inulcerative colitis. Br MedJ' 1965;i:827-31.

7Jankey N, Price LA. Small intestinal histochemical and histologicalchanges in ulcerative colitis. Gut 1969;10:267-9.

8 Binder V, Soltoft J, Gudmond-Hoyer E. Histological and histochemicalchanges in the jejunal mucosa in ulcerative colitis. ScandJ7 Gastroenterol1974;9 :293-7.

9 Read NW, Krejs GJ, Read MG, Santa Ana CA, Morawski SG, FordtranJS. Chronic diarrhoea of unknown origin. Gastroenterology 1980;78:264-71.

10 Morson BC. Histopathology of cathartic colon. Gut 1971 ;12:867-8.11 Smith B. Pathology of cathartic colon. Proc R Soc Med 1972;65:288.

(Accepted 5 August 1982)

Rate of inactivation of cytomegalovirus in raw banked milkduring storage at -200C and pasteurisation

HENRIK FRIIS, H KERZEL ANDERSEN

Abstract

Samples of milk from 23 mothers attending the depart-ment of obstetrics and gynaecology and 36 who donatedmilk to the department's milk bank were cultured forcytomegalovirus. Virus was isolated from samples from12 of the milk donors but none of the mothers attendingthe department; follow-up studies during lactation inseven of these 12 women showed that five continued toexcrete the virus. Samples were taken on three occasionsfrom one woman who regularly excreted high titres of thevirus. Storage at -20°C for over three days reduced thetitre by over 99%; after pasteurisation at 63°C for eightminutes the milk did not contain any viable virus.

It is recommended that raw banked milk used for feed-ing preterm babies should be kept frozen for at least 72hours before feeding.

Introduction

Recently it has been questioned whether human milk used to feedinfants who cannot be breast-fed should be pasteurised or notbecause pasteurisation affects antimicrobial substances andnutritional properties of milk.1 2 Little attention, however, hasbeen paid to the risk of transmitting cytomegalovirus infectionby using raw banked milk. For this reason we studied theamounts of cytomegalovirus in human milk and the rate of

Institute of Medical Microbiology, University of Aarhus, DK-8000Aarhus C, Denmark

HENRIK FRIIS, medical studentH KERZEL ANDERSEN, DMSC, reader in virology

inactivation of the virus during storage at - 20°C and duringpasteurisation at 63°C.

Patients, methods, and results

Milk samples were collected between days 2 and 12 from 23 mothersattending the university department of obstetrics and gynaecologyat Aarhus Kommunehospital, and from 36 women who donated milkto the milk department's milk bank. Within four hours after collection1 ml of skimmed milk was inoculated into a 50 cm2 monolayer ofhuman fibroblasts in glass bottles. After one hour of adsorption thecultures were overlaid with Eagle's modified medium and studiedweekly for four weeks for a cytopathic effect typical for cytomegalo-virus. Titres of cytomegalovirus were estimated by counting viralplaques in the cell layer after staining with methylene blue as describedpreviously.3 Cytomegalovirus was not isolated from any samples fromthe 23 mothers attending the department but from 45 samples from 12of the 36 milk donors. The earliest isolation was made in milk collectedon day 14 and the latest in milk collected 58 weeks after delivery.The initial cytomegalovirus titres were about 1000 plaque-forming

units (pfu)/ml in two women, 500 and 200 pfu/ml in two others,and about 10 pfu/ml in the rest. Follow-up studies performed duringlactation in seven of the milk donors including three of the four womenwith initial titres of 200 pfu or more/ml showed that all except twocontinued to excrete cytomegalovirus, in titres of 10 pfu or less/ml.One woman, however, regularly excreted between 1000 and 4600pfu/ml for 26 weeks of lactation; on three occasions samples obtainedfrom her were divided and cultured after storage at - 20°C for up to10 days. Storage for more than three days reduced the amount of cyto-megalovirus in the milk by more than 99°, (from 4300 pfu/ml toabout 10 pfu/ml) (figure). Pasteurisation at 63°C, by heating aliquotsof milk in a water bath for one, two, four, eight, and 16 minutes fol-lowed by quick cooling in an ice bath, also reduced the virus titre(from 3600 pfu/ml to 10 pfu/ml after one minute). After eight minutesof pasteurisation the milk did not contain any viable cytomegalovirusat all.

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